October

From 2008.igem.org

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__NOTOC__
__NOTOC__
 +
 +
<h3>Oct. 1st 2008</h3><br>
 +
'''Digestion transfectionvector and CMV PCR product(3rd try)'''(Kathrin)<br>
 +
Digestion with EcoRI and PstI<br>
 +
<br>
 +
'''Ligation transfectionvector and CMV PCR product(3rd try)''' (Kathrin)<br>
 +
Using new T4 DNA ligase and new ligase buffer<br>
 +
<br>
 +
'''Miniprep of pBSK-signalpeptide''' (Sabine)<br>
 +
<br>
 +
'''Transformation of pMA-transmembrane, pMA-Histag, pMA-signalpeptide, pMA-splitlinker, pMA-StreptagII''' (Sabine)<br>
 +
<br>
 +
 +
<h3>Oct. 2nd 2008</h3><br>
 +
'''Picking clones''' (Kathrin)<br>
 +
pMA-transmembrane, pMA-Histag, pMA-signalpeptide, pMA-splitlinker, pMA-StreptagII<br>
 +
<br>
 +
'''Digestion of:''' (Kathrin)<br>
 +
pBSK-signalpeptide with AgeI and PstI<br>
 +
pMA-Lipocalin and pMA-Anti-Nip-scFv with NgoMIV and PstI<br>
 +
<br>
 +
'''Ligation''' (Kathrin)<br>
 +
Using Quick DNA ligase<br>
 +
Vector: pBSK-signalpeptide<br>
 +
Insert: Lipocalin, Anti-NIP-scFv<br>
 +
-Approaches: Insert/Vector -> a) 6/2, b) 4/4<br>
 +
<br>
 +
'''Transformation''' (Kathrin)<br>
 +
of the ligation transfectionvector+CMV PCR product(3rd try)<br>
 +
<br>
 +
'''Miniprep of:''' (Sabine)<br>
 +
pMA-transmembrane, pMA-Histag, pMA-signalpeptide, pMA-splitlinker, pMA-StreptagII<br>
 +
<br>
 +
'''Preparative digestion of:''' (Sabine)<br>
 +
- pMA-splitlinker with AgeI and SpeI<br>
 +
- pMA-Cerulan split C-CFP and pMA-Venus split C-GFP with NgoMIV and SpeI<br>
 +
<br>
 +
'''Ligation''' (Sabine)<br>
 +
Vector: pMA-splitlinker<br>
 +
Insert: Cerulan split C-CFP, Venus split C-GFP<br>
 +
<br>
 +
'''Transformation''' (Sabine)<br>
 +
of the ligation pBSK-signalpeptide+Lipocalin and pBSK-signalpeptide+Anti-NIP-scFv<br><br>
 +
<h3>Oct. 3rd 2008</h3><br>
 +
'''Transformation''' (Michael)<br>
 +
of the ligation pMA-splitlinker+Cerulan split C-CFP and pMA-splitlinker+Venus split C-GFP<br>
 +
<br>
 +
'''Picking clones''' (Michael)<br>
 +
-transfectionvector-CMV PCR product<br>
 +
-pBSK-signalpeptide-Lipocalin<br>
 +
-pBSK-signalpeptide-Anti-NIP-scFv<br>
 +
<br>
 +
'''Miniprep and analytic digestion of''' (Kathrin)<br>
 +
-transfectionvector-CMV PCR product<br>
 +
-pBSK-signalpeptide-Lipocalin<br>
 +
-pBSK-signalpeptide-Anti-NIP-scFv<br>
 +
<br>
 +
'''Preparative digestion of''' (Kathrin)<br>
 +
-transfectionvector-CMV PCR product with SpeI and PstI<br>
 +
-pBSK-signalpeptide-Lipocalin and pBSK-signalpeptide-Anti-NIP-scFv with XbaI and PstI<br>
 +
<br>
 +
'''Ligation''' (Sabine)<br>
 +
Vector: transfectionvector-CMV PCR product<br>
 +
Insert: signalpeptide-Lipocalin, signalpeptide-Anti-NIP-scFv<br>
 +
<br>
 +
<h3>Oct. 4th 2008</h3><br>
 +
<br>
 +
'''Transformation''' (Normann)<br>
 +
-the ligation transfectionvector-CMV PCR product+signalpeptide-Lipocalin<br>
 +
-the ligation transfectionvector-CMV PCR product+signalpeptide-Anti-NIP-scFv<br>
 +
-pGA18-bla1(ß-Lactamase 1)<br>
 +
-pGA18-bla2(ß-Lactamase 2)<br>
 +
-pGA14-YFP<br>
 +
<br>
 +
'''Picking clones''' (Sabine)<br>
 +
-pMA-splitlinker-Cerulan split C-CFP<br>
 +
-pMA-splitlinker-Venus split C-GFP<br>
 +
<br>
 +
<h3>Oct. 5th 2008</h3><br>
 +
<br>
 +
'''Miniprep of:''' (Michael)<br>
 +
-pMA-splitlinker-Cerulan split C-CFP<br>
 +
-pMA-splitlinker-Venus split C-GFP<br>
 +
-pGA18-bla1<br>
 +
-pGA18-bla2<br>
 +
-pGA14-YFP<br>
 +
<br>
 +
'''Picking clones''' (Sabine)<br>
 +
-transfectionvector-CMV PCR product-signalpeptide-Lipocalin<br>
 +
-transfectionvector-CMV PCR product-signalpeptide-Anti-NIP-scFv<br>
 +
<br>
 +
'''Analytic digestion of:''' (Sabine)<br>
 +
-pMA-splitlinker-Cerulan split C-CFP -> 3 correct clones<br>
 +
-pMA-splitlinker-Venus split C-GFP -> 2 correct clones<br>
 +
<br>
 +
'''Preparative digestion''' (Sabine)<br>
 +
-pGA14-YFP<br>
 +
<br>
 +
'''Ligation''' (Sabine)<br>
 +
Using T4 DNA ligase<br>
 +
Vector: transfectionvector-CMV PCR product<br>
 +
Insert: YFP<br>
 +
<br>
 +
<h3>Oct. 6th 2008</h3>
 +
<br>
 +
'''DNA-Origamis''' normann <br>
 +
DNA-Origamis with Alexa/noNIP, NIP/Alexa, NIP, noNIP were made.
 +
The protocol of july 24th was used.<br>
 +
<br>
 +
'''Preparation of 293T transfection''' normann<br>
 +
<br> In order to transfect the 293T-cells  with CMV+YFP tomorrow, the amount of cells per ml was determined with the "Neubauer cell chamber" --> 2,62*10E6 cells/ml <br>
 +
To transfect cells with 1µg DNA in a 6-well plate, you need ~6*10E4 cells per well, so 20µl of the suspension was given in each well and 2ml DMEM was added.<br>
 +
<br>
 +
'''Transformation of the ligation''' (Sabine)<br>
 +
transfectionvector-CMV PCR product+YFP<br>
 +
<br>
 +
'''Preparative digestion of''' (Sabine)<br>
 +
-pMA-transmembrane with AgeI and SpeI<br>
 +
-pMA-Cerulan N-CFP and pMA-Venus N-GFP and NgoMIV and SpeI<br>
 +
<br>
 +
'''Preparative digestion of''' (Kathrin)<br>
 +
-pMA-splitlinker-Cerulan split C-CFP<br>
 +
-pMA-splitlinker-Venus split C-GFP<br>
 +
<br>
 +
'''Analytic digestion of''' (Kathrin)<br>
 +
-transfectionvector-CMV PCR product-signalpeptide-Lipocalin -> no correct clones<br>
 +
-transfectionvector-CMV PCR product-signalpeptide-Anti-NIP-scFv -> no correct clones<br>
 +
'''Ligation''' (Michael)<br>
 +
Using Quick ligase<br>
 +
Vector: pMA-transmembrane<br>
 +
Insert: splitlinker-Cerulan split C-CFP, splitlinker-Venus split C-GFP, Cerulan N-CFP and Venus N-GFP<br>
 +
<br>
 +
'''Transformation of the ligation''' (Michael)<br>
 +
-pMA-transmembrane+splitlinker-Cerulan split C-CFP<br>
 +
-pMA-transmembrane+splitlinker-Venus split C-GFP<br>
 +
-pMA-transmembrane+Cerulan N-CFP<br>
 +
-pMA-transmembrane+Venus N-GFP<br>
 +
<br>
 +
'''Picking clones''' (Michael)<br>
 +
-transfectionvector-CMV PCR product-YFP<br>
 +
<br>
 +
<h3>Oct. 7th 2008</h3>
 +
<br>
 +
'''Miniprep and analytic digestion''' (Kathrin)<br>
 +
- transfectionvector-CMV PCR product-YFP clones 1-4<br>
 +
- transfectionvector-CMV PCR product-Lipocalin clones 5-7<br>
 +
<br>
 +
test digestion of the clones named above with NotI<br>
 +
analysis on the agarosegel shows not the expected bands
 +
 +
<h3>Oct. 8th 2008</h3> <br>
 +
'''Digestion of CFP''' normann<br>
 +
In order to put the gene for CFP behind the CMV-promotor on our transfectionvector to make a double trasnsfektion with YFP, the plasmit carrieng CFP was digested using pst1 and Xba1. <br>
 +
The digested gene was brought on an agarosegel and the expected band was cut out, after the followed the extraction out of the gel using the "QIAquick gel extraction kit"<br>
 +
<br>
 +
'''preparation of ss Phage DNA''' normann <br>
 +
Because the ss PhageDNA ,which is needed for the origamis, is nearly empty phages were harvested out of a overnight culture. <br>
 +
Harvesting happened like it is descriped under Methods. <br>
 +
Preparation of the ssDNA was done following the QIAPrep Spin M13 Kits protocol. <br>
 +
<br>
 +
'''Klenow-fill-in-reaction''' (Kathrin)<br>
 +
For producing a GGGS-Linker<br>
 +
<br>
 +
'''Preparative digestion''' (Sabine)<br>
 +
-pMA-transmembrane with AgeI and SpeI<br>
 +
-pMA-BB058(Split luciferase), pGA18-bla1, pGA18-bla2 with NgoMIV and SpeI<br>
 +
<br>
 +
'''Ligation''' (Sabine)<br>
 +
Using Quick ligase<br>
 +
Vector: pMA-transmembrane<br>
 +
Insert: BB058(Split luciferase), bla1 and bla2<br>
 +
<br>
 +
'''Transformation''' (Sabine)<br>
 +
of the ligations<br>
 +
-pMA-transmembrane+BB058(Split luciferase)<br>
 +
-pMA-transmembrane+bla1<br>
 +
-pMA-transmembrane+bla2<br>
 +
<br>
 +
'''Miniprep''' (Sabine)<br>
 +
-transfectionvector-CMV PCR product clones 5-9<br>
 +
<br>
 +
<h3>Oct. 9th 2008</h3> <br>
 +
'''Analytic digestion (with NotI)''' (Kathrin)<br>
 +
-transfectionvector-CMV PCR product clones 5-9 -> correct clones<br>
 +
<br>
 +
'''Preparative digestion''' (Kathrin)<br>
 +
-transfectionvector-CMV PCR product clone 5 with SpeI and PstI<br>
 +
-pBSK-signalpeptide-Lipocalin and pBSK-signalpeptide-Anti-Nip-sc-Fv with XbaI and PstI<br>
 +
<br>
 +
'''Sequencing''' (Kathrin)<br>
 +
Correct:<br>
 +
-pMA-transmembrane-Cerulan-N-CFP clone 2<br>
 +
-pMA-transmembrane-Venus-N-GFP clone 3<br>
 +
-pMA-transmembrane-splitlinker-Cerulan-C-CFP clone 2<br>
 +
-pMA-transmembrane-splitlinker-Venus-C-GFP clone 3<br>
 +
Not correct:<br>
 +
-pBSK-signalpeptide-Lipocalin clone 1<br>
 +
-pBSK-signalpeptide-Anti-NIP-sc-Fv clone 4<br>
 +
<br>
 +
 +
<h3>Oct. 11th 2008</h3> <br>
 +
'''Ligation of CFP/YFP and Transfectionvektor+CMV'''
 +
<br> Ligation was done using the quick ligase. Inkubation for 40min.
 +
<br>
 +
<br>'''Transformation of TV-CMV-YFP and TV-CMV-CFP'''
 +
}}
}}

Revision as of 15:45, 19 October 2008


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__october




Oct. 1st 2008


Digestion transfectionvector and CMV PCR product(3rd try)(Kathrin)
Digestion with EcoRI and PstI

Ligation transfectionvector and CMV PCR product(3rd try) (Kathrin)
Using new T4 DNA ligase and new ligase buffer

Miniprep of pBSK-signalpeptide (Sabine)

Transformation of pMA-transmembrane, pMA-Histag, pMA-signalpeptide, pMA-splitlinker, pMA-StreptagII (Sabine)

Oct. 2nd 2008


Picking clones (Kathrin)
pMA-transmembrane, pMA-Histag, pMA-signalpeptide, pMA-splitlinker, pMA-StreptagII

Digestion of: (Kathrin)
pBSK-signalpeptide with AgeI and PstI
pMA-Lipocalin and pMA-Anti-Nip-scFv with NgoMIV and PstI

Ligation (Kathrin)
Using Quick DNA ligase
Vector: pBSK-signalpeptide
Insert: Lipocalin, Anti-NIP-scFv
-Approaches: Insert/Vector -> a) 6/2, b) 4/4

Transformation (Kathrin)
of the ligation transfectionvector+CMV PCR product(3rd try)

Miniprep of: (Sabine)
pMA-transmembrane, pMA-Histag, pMA-signalpeptide, pMA-splitlinker, pMA-StreptagII

Preparative digestion of: (Sabine)
- pMA-splitlinker with AgeI and SpeI
- pMA-Cerulan split C-CFP and pMA-Venus split C-GFP with NgoMIV and SpeI

Ligation (Sabine)
Vector: pMA-splitlinker
Insert: Cerulan split C-CFP, Venus split C-GFP

Transformation (Sabine)
of the ligation pBSK-signalpeptide+Lipocalin and pBSK-signalpeptide+Anti-NIP-scFv

Oct. 3rd 2008


Transformation (Michael)
of the ligation pMA-splitlinker+Cerulan split C-CFP and pMA-splitlinker+Venus split C-GFP

Picking clones (Michael)
-transfectionvector-CMV PCR product
-pBSK-signalpeptide-Lipocalin
-pBSK-signalpeptide-Anti-NIP-scFv

Miniprep and analytic digestion of (Kathrin)
-transfectionvector-CMV PCR product
-pBSK-signalpeptide-Lipocalin
-pBSK-signalpeptide-Anti-NIP-scFv

Preparative digestion of (Kathrin)
-transfectionvector-CMV PCR product with SpeI and PstI
-pBSK-signalpeptide-Lipocalin and pBSK-signalpeptide-Anti-NIP-scFv with XbaI and PstI

Ligation (Sabine)
Vector: transfectionvector-CMV PCR product
Insert: signalpeptide-Lipocalin, signalpeptide-Anti-NIP-scFv

Oct. 4th 2008



Transformation (Normann)
-the ligation transfectionvector-CMV PCR product+signalpeptide-Lipocalin
-the ligation transfectionvector-CMV PCR product+signalpeptide-Anti-NIP-scFv
-pGA18-bla1(ß-Lactamase 1)
-pGA18-bla2(ß-Lactamase 2)
-pGA14-YFP

Picking clones (Sabine)
-pMA-splitlinker-Cerulan split C-CFP
-pMA-splitlinker-Venus split C-GFP

Oct. 5th 2008



Miniprep of: (Michael)
-pMA-splitlinker-Cerulan split C-CFP
-pMA-splitlinker-Venus split C-GFP
-pGA18-bla1
-pGA18-bla2
-pGA14-YFP

Picking clones (Sabine)
-transfectionvector-CMV PCR product-signalpeptide-Lipocalin
-transfectionvector-CMV PCR product-signalpeptide-Anti-NIP-scFv

Analytic digestion of: (Sabine)
-pMA-splitlinker-Cerulan split C-CFP -> 3 correct clones
-pMA-splitlinker-Venus split C-GFP -> 2 correct clones

Preparative digestion (Sabine)
-pGA14-YFP

Ligation (Sabine)
Using T4 DNA ligase
Vector: transfectionvector-CMV PCR product
Insert: YFP

Oct. 6th 2008


DNA-Origamis normann
DNA-Origamis with Alexa/noNIP, NIP/Alexa, NIP, noNIP were made. The protocol of july 24th was used.

Preparation of 293T transfection normann

In order to transfect the 293T-cells with CMV+YFP tomorrow, the amount of cells per ml was determined with the "Neubauer cell chamber" --> 2,62*10E6 cells/ml
To transfect cells with 1µg DNA in a 6-well plate, you need ~6*10E4 cells per well, so 20µl of the suspension was given in each well and 2ml DMEM was added.

Transformation of the ligation (Sabine)
transfectionvector-CMV PCR product+YFP

Preparative digestion of (Sabine)
-pMA-transmembrane with AgeI and SpeI
-pMA-Cerulan N-CFP and pMA-Venus N-GFP and NgoMIV and SpeI

Preparative digestion of (Kathrin)
-pMA-splitlinker-Cerulan split C-CFP
-pMA-splitlinker-Venus split C-GFP

Analytic digestion of (Kathrin)
-transfectionvector-CMV PCR product-signalpeptide-Lipocalin -> no correct clones
-transfectionvector-CMV PCR product-signalpeptide-Anti-NIP-scFv -> no correct clones
Ligation (Michael)
Using Quick ligase
Vector: pMA-transmembrane
Insert: splitlinker-Cerulan split C-CFP, splitlinker-Venus split C-GFP, Cerulan N-CFP and Venus N-GFP

Transformation of the ligation (Michael)
-pMA-transmembrane+splitlinker-Cerulan split C-CFP
-pMA-transmembrane+splitlinker-Venus split C-GFP
-pMA-transmembrane+Cerulan N-CFP
-pMA-transmembrane+Venus N-GFP

Picking clones (Michael)
-transfectionvector-CMV PCR product-YFP

Oct. 7th 2008


Miniprep and analytic digestion (Kathrin)
- transfectionvector-CMV PCR product-YFP clones 1-4
- transfectionvector-CMV PCR product-Lipocalin clones 5-7

test digestion of the clones named above with NotI
analysis on the agarosegel shows not the expected bands

Oct. 8th 2008


Digestion of CFP normann
In order to put the gene for CFP behind the CMV-promotor on our transfectionvector to make a double trasnsfektion with YFP, the plasmit carrieng CFP was digested using pst1 and Xba1.
The digested gene was brought on an agarosegel and the expected band was cut out, after the followed the extraction out of the gel using the "QIAquick gel extraction kit"

preparation of ss Phage DNA normann
Because the ss PhageDNA ,which is needed for the origamis, is nearly empty phages were harvested out of a overnight culture.
Harvesting happened like it is descriped under Methods.
Preparation of the ssDNA was done following the QIAPrep Spin M13 Kits protocol.

Klenow-fill-in-reaction (Kathrin)
For producing a GGGS-Linker

Preparative digestion (Sabine)
-pMA-transmembrane with AgeI and SpeI
-pMA-BB058(Split luciferase), pGA18-bla1, pGA18-bla2 with NgoMIV and SpeI

Ligation (Sabine)
Using Quick ligase
Vector: pMA-transmembrane
Insert: BB058(Split luciferase), bla1 and bla2

Transformation (Sabine)
of the ligations
-pMA-transmembrane+BB058(Split luciferase)
-pMA-transmembrane+bla1
-pMA-transmembrane+bla2

Miniprep (Sabine)
-transfectionvector-CMV PCR product clones 5-9

Oct. 9th 2008


Analytic digestion (with NotI) (Kathrin)
-transfectionvector-CMV PCR product clones 5-9 -> correct clones

Preparative digestion (Kathrin)
-transfectionvector-CMV PCR product clone 5 with SpeI and PstI
-pBSK-signalpeptide-Lipocalin and pBSK-signalpeptide-Anti-Nip-sc-Fv with XbaI and PstI

Sequencing (Kathrin)
Correct:
-pMA-transmembrane-Cerulan-N-CFP clone 2
-pMA-transmembrane-Venus-N-GFP clone 3
-pMA-transmembrane-splitlinker-Cerulan-C-CFP clone 2
-pMA-transmembrane-splitlinker-Venus-C-GFP clone 3
Not correct:
-pBSK-signalpeptide-Lipocalin clone 1
-pBSK-signalpeptide-Anti-NIP-sc-Fv clone 4

Oct. 11th 2008


Ligation of CFP/YFP and Transfectionvektor+CMV
Ligation was done using the quick ligase. Inkubation for 40min.

Transformation of TV-CMV-YFP and TV-CMV-CFP

Freiburg08 FT3.png

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